O^-Alkylguanine-DNA alkyltransferase (AGT) is an important DMArepair protein that is a major resistance mechanism protecting from the carcinogenic, cytotoxic and mutagenic effects of alkylating agents including nitrosamines and nitrosamides, which are important human carcinogens. It acts to repair O^-alkylguanine adducts by transferring the alkyl group to a cysteine acceptor residue in the protein. The proposed experiments are focussed on the properties of AGT and related proteins and aimed at providing a more complete understanding of the manner by which damage to DNA is influenced by these proteins. The planned investigations build on advances based on studies carried out in the previous period of support. These include: the description of a model for the mechanism of the AGT reaction based on structural studies and the properties of AGT mutants generated by site-directed mutagenesis; studies of the substrate specificity, kinetics and directionality of repair by AGT; investigation of the interactions of AGT with itself in the presence of DNA and with other proteins; the characterization of several proteins related to AGT that also protect against alkylation damage; and studies showing the mechanism by which AGT paradoxically enhances the genotoxicity of dihalplalkanes. Most of the experimental techniques and reagents needed including mutant AGT proteins, vectors for expressing them and the AGT like proteins and cells expressing them are already available in the Pis laboratory and the proposed experiments are a logical extension of the ongoing work. There are 4 interrelated specific aims: (1) To study the mechanism and importance of the paradoxical AGT-mediated increase in DNA damage caused by dihalohaloalkanes and other bifunctional agents. (2) To continue studies on a fusion protein that we have found in a microorganism that lives under extreme conditions what has both AGT and EndoV activity. (3) To investigate the function of ALS (alkyltransferase like sequence), a novel protein that binds to O6- alkylguanine and protects from alkylation damage but does not by itself repair these adducts. (4) To investigate the mechanism of AGT repair of DNA and variations in repair by polymorphic variants of hAGT. These studies will focus on understanding how AGT finds and recognizes lesions and how the inactivated alkylated form of AGT is disposed of. These experiments will aid in the understanding of the individual risks associated with exposure to alkylating agents and an important class of environmental carcinogens.